A cargo de-ubiquitination switch in membrane remodeling in the endocytic pathways.
Ubiquitination controls the regulation of cell surface proteins, required for cellular homeostasis in eukaryotic cells. When cell surface proteins need to be down-regulated, one or several ubiquitin moieties are covalently, but reversibly attached to cell surface proteins to serve as signal for their internalization and sorting into recycling or degradation pathways. Ubiquitinated cargo destined for the degradative pathway recruits the endosomal sorting complexes required for transport (ESCRT)-0, -I, -II and -III/Vps4. These encapsulate the cargo into endosomal intraluminal vesicles (ILVs), resulting in multivesicular endosomes that fuse with the vacuole/lysosome for cargo degradation. The molecular mechanism by which ILV membranes are severed and released inside MVBs isn’t fully understood but includes a dynamic recruitment cascade of different ESCRT-III subunits, mediated by the AAA ATPase Vps4. Ubiquitin on cargo molecules about to be internalized by the ESCRT-III/Vps4 inhibits this cascade, and the de-ubiquitination by Doa4 relieves this inhibition. We hypothesize that Doa4-mediated de-ubiquitination serves as an on-switch for ESCRT-III-mediated ILV internalization and want to engineer this switch into a tool that allows us to gain control of the timing of ESCRT-III/Vps4-mediated membrane remodeling events.
We have previously established assays that allow for reconstituting different aspects of the Vps4-mediated ESCRT-III dynamics1-3. Here, we propose to test in vitro as to whether Doa4 binds both early ESCRTs and ubiquitin by coincidence detection. We hypothesize that Doa4 then removes ubiquitin through its de-ubiquitination activity, serving as an on-switch for ESCRT-III dynamics. We will investigate the ultrastructure of the interaction between Doa4 and the ESCRT-III machinery using EM. To this end, we will reconstitute the Doa4-bound ESCRT-III on membranes to elucidate how their interaction affects ESCRT-III assemblies. Ultimately, we want to use the Doa4 DUB on-switch to synchronize ESCRT-III dynamics at sites of ILV formation in cells, allowing us to gain further insight into the mechanisms by which the ESCRT-III can sever cellular membranes.
Dr. Joachim Moser von Filsek (BZH Heidelberg)